Mouritzen Michelle V, Abourayale Sali, Ejaz Rooshanie, Ardon Christine B, Carvalho Eugenia, Dalgaard Louise T, Roursgaard Martin, Jenssen Håvard
Department of Science and Environment, Roskilde University, Roskilde, Denmark.
Department of Dermatology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, Netherlands.
J Pept Sci. 2018 Jul;24(7):e3093. doi: 10.1002/psc.3093. Epub 2018 Jun 25.
Neurotensin, substance P, and insulin have been demonstrated to improve wound healing in vivo. However, the mechanism behind their effect is still not fully understood. This study investigates the effects leading to enhanced scratch closure by these peptides in vitro. The skin keratinocyte cell line, HaCaT, was used to test scratch closure effects of the peptides and alterations of cytokine levels. HUVEC cells were used to test the angiogenic effect of the peptides. Furthermore, clinical isolates of Staphylococcus lugdunensis were used to examine the potential antimicrobial activity of each peptide. Our results demonstrate that neurotensin, substance P, and insulin had significant migratory effects in scratch assays were neurotensin had the lowest effect. Furthermore, we investigated use of the peptides in combination. When substance P was used in combination with neurotensin, the cell migratory capacity was decreased, and the peptides showed a negative correlation (r = -0.298, P < .001). Neurotensin and insulin significantly increased levels of monocyte chemoattractant protein-1 (P < .001) secreted from white blood cells, whereas substance P showed a tendency. Interestingly, neurotensin increased the level of monocyte chemoattractant protein-1 significantly compared to substance P (P < .01). Additionally, the peptides decreased TNFα mRNA levels (P < .001) in HaCaT cells, whereas only neurotensin and insulin decreased IL-8 mRNA (P < .001) but had no significant effect on IL-6 mRNA levels. Surprisingly, substance P increased IL-6 mRNA 9-fold (P < .001). Furthermore, we demonstrate that the peptides increased angiogenesis in the HUVEC cells (P < .001). Finally, S. lugdunensis isolates were not susceptible to the peptides. We demonstrate that the peptides worked differently on HaCaT cells, but substance P acted differently than neurotensin on cytokine levels expression as well as on migration of HaCaT cells. On the contrary, neurotensin and insulin worked similarly. All of these aspects are crucial for proper wound healing, and the results suggest multiple mechanisms for wound-healing properties of these peptides.
神经降压素、P物质和胰岛素已被证明在体内可促进伤口愈合。然而，它们作用背后的机制仍未完全明确。本研究在体外探究了这些肽导致划痕闭合增强的作用。使用皮肤角质形成细胞系HaCaT来测试这些肽的划痕闭合效果以及细胞因子水平的变化。使用人脐静脉内皮细胞（HUVEC）来测试这些肽的血管生成作用。此外，使用路邓葡萄球菌的临床分离株来检测每种肽的潜在抗菌活性。我们的结果表明，神经降压素、P物质和胰岛素在划痕试验中具有显著的迁移作用，其中神经降压素的作用最小。此外，我们研究了肽的联合使用情况。当P物质与神经降压素联合使用时，细胞迁移能力下降，且这些肽呈现负相关（r = -0.298，P <.001）。神经降压素和胰岛素显著提高了白细胞分泌的单核细胞趋化蛋白-1水平（P <.001），而P物质表现出一定趋势。有趣的是，与P物质相比，神经降压素显著提高了单核细胞趋化蛋白-1水平（P <.01）。此外，这些肽降低了HaCaT细胞中TNFα mRNA水平（P <.001），而只有神经降压素和胰岛素降低了IL-8 mRNA水平（P <.001），但对IL-6 mRNA水平无显著影响。令人惊讶的是，P物质使IL-6 mRNA增加了9倍（P <.001）。此外，我们证明这些肽增加了HUVEC细胞中的血管生成（P <.001）。最后，路邓葡萄球菌分离株对这些肽不敏感。我们证明这些肽对HaCaT细胞的作用方式不同，但P物质在细胞因子水平表达以及HaCaT细胞迁移方面的作用与神经降压素不同。相反，神经降压素和胰岛素的作用相似。所有这些方面对于伤口的正常愈合都至关重要，结果表明这些肽的伤口愈合特性存在多种机制。
